Date pit composition for the treatment of animals

ABSTRACT

A method of treating or preventing bacterial infection in an animal comprises administering an effective amount of degraded date pits. Degraded date pits can be produced by the treatment of date pits with fungi.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No.14/614,113, filed Feb. 4, 2015, which is a divisional of U.S.application Ser. No. 13/284,173, filed Oct. 28, 2011, now U.S. Pat. No.8,968,729, both of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates to compositions for treating and preventingbacterial infections in an animal. In particular the invention relatesto compositions comprising degraded date pits.

BACKGROUND OF THE INVENTION

Sub-therapeutic doses of antibiotics have routinely been administered toanimals in order to promote weight gain in apparently healthy animals.While supplementing animal feed with antibiotics can have a number ofbenefits, concerns exist over the use of conventional antibiotics inanimal feed and water. The use of antibiotics in sub-therapeutic levelsin animals has been implicated in the rise in antibiotic resistance ofbacteria. Additionally, residual antibiotics may remain in meat productsthat are meant for human consumption.

To address these concerns the US Food and Drug agency (FDA) require thatthat antibiotic must be withdrawn from the feed of the animal at leasttwo weeks prior to slaughter to prevent antibiotics remaining in theanimal that is to enter the human food chain. The European Union andother countries require that antibiotics are not used as growthpromoters in animal feed. Furthermore feed composition costs make alarge proportion of the costs in animal production.

Date pits are readily available in a number of countries. Date pits havetypically been seen as waste product from the preparation of dates andare usually discarded.

SUMMARY OF THE INVENTION

An object of the invention is to provide methods and compositions totreat and prevent bacterial infections in domestic animals.

A further object of the invention is to provide an alternative toconventional antibiotics to treat and prevent bacterial infections indomestic animals.

In one aspect, the invention comprises a method of treating andpreventing bacterial infection in animals comprising: administering aneffective amount of a degraded date pit composition to the animal.

The method includes administering the degraded date pit composition inan effective amount thereby preventing or delaying the onset of abacterial infection in the animal, or reducing the microbial load in theanimal or inhibiting the growth or killing bacterium in the animal. Themethods allow a reduction in the amount of conventional antibiotics thatwill need to be administered to maintain animal health.

The degraded date pit composition can be administered as a component ofan animal feed composition. The animal feed can comprise between 5-20%(w/w) of a degraded date pit composition. Preferably the animal feed cancomprise approximately 10% (w/w) of a degraded date pit composition.

The degraded date pit composition can comprise mannan-oligosaccharides(MOS) and/or comprise free mannose.

Bacterial infection may be caused by Salmonella, Campylobacter, Shigellaor Escherichia coli.

The method can be used for treating an animal selected from the groupconsisting of cattle, horses, pigs, goats, fish and poultry. Preferablythe animal is poultry.

A further aspect of the invention comprises an antibacterial feedadditive for animals comprising degraded date pits.

The degraded date pits can be fungi treated date pits.

The antibacterial feed additive can be in the form of a powder.

A further aspect of the invention comprises an animal feed compositioncomprising the antibacterial feed additive as described. The degradeddate pits provide an inexpensive antibacterial additive in use in ananimal feed composition.

The animal feed comprises 5-20% (w/w) of the feed additive. Preferablythe animal feed can comprise 10% (w/w) of the feed additive.

The animal feed can be a soy-corn based feed. The animal feed maycomprise a blend of corn, soybean meal, vitamins, minerals, amino acids,corn oil and fish meal.

A further aspect of the invention comprises a method of producing anantibacterial feed stuff for treating and preventing bacterial infectionin animals, the method comprising: treating date pits with a fungus toform a degraded date pit composition; and mixing the degraded date pitcomposition with an animal feed.

The degraded date pit composition can be mixed into the feed compositionat a concentration of 5-20% (w/w). The degraded date pit composition canbe mixed in the feed composition at a concentration of 10% (w/w).

Treating the date pits with a bacteria can comprise incubating the datepits and fungus together. The fungus used to treat the bacteria can beTrichoderma reesei.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a schematic view of a Solid State Degradation system.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises methods and compositions for treating andpreventing bacterial infections in animals. The composition comprisesdegraded date pits which are administered to animals in an effectiveamount to treat or prevent bacterial infection. In some embodiments theanimal will already have an infection and the degraded date pitcomposition is administered to treat the bacterial infection or reducethe bacterial load. In other embodiments the animal may not have aninfection e.g. the degraded date pit composition is administered toprevent or delay the onset of a bacterial infection in the animal.

The term “degraded date pit” refers to a composition wherein date pitshave been treated such that dietary fibres in the date pits are brokendown into their digestible units.

One method for obtaining degraded date pit comprises treating date pitswith a fungus to obtain a degraded date pit composition. A fungussuitable for degrading date pits is Trichoerma reesei. Other fungus,such as yeast, and which are capable of breaking down indigestiblefibres found in date pits into digestible units can also be used.Trichoerma reesei can degrade date pits by breaking down mannan fibreinto different products such as free mannose and mannan-oligosacchrides(MOS). Other fibres that Trichoerma reesei can break down into theirdigestible units include lignin, cellulose and hemicelluloses.

The date pits can be treated within a solid state degradation system byincubating the fresh dates in the presence of Trichoerma reesei underconditions suitable to promote degradation of the date pits. A solidstate degradation system 10 as exemplified in FIG. 1 can be used toprocess the date pits. The date pits and fungi 12 are layered within aseries of cones 14. Each cone 14 is closed by a cover 16, to avoidcontamination. Preferably the cover is a fibre glass cover. Duringincubation air is blown from an air supply 18 into a water tank 20. Themoist air created is then supplied to the cones 14. A disinfection unit22 treats the moist air before it is supplied to the cones.

Once a degraded date pit composition is obtained it can be mixed withconventional animal feed ingredients to produce a supplemented animalfeed composition having antibacterial properties.

The term “feed composition”, refers to a preparation providingnutritional value to the animal. Preferred animals include cattle, pigs,goats, sheep, fish, horses, and poultry such as chickens, turkey, ducks,geese, ostriches, quails, pheasants or other domestic fowl. Preferablythe animal being treated is poultry.

Any conventional animal feed composition may be supplemented with thedegraded date pit composition. An animal feed can comprise a blend ofcorn, soybean meal, limestone, salt, dicalcium phosphate, vitamins,minerals, amino acids (such as DL-methionine and lysine), corn oil, fishmeal. The feed composition can comprise further components such asfurther amino acids, enzymes and other nutritional components. The feedcomposition can be prepared by mixing the ingredients together in therequired portions. The feed composition can comprise different portionsof ingredients depending on the age of the animal the composition isbeing fed to. Thereby, having different feed compositions administeredat different stages of the animal's life.

The degraded date pits can be added to the feed composition during themanufacture of the feed composition. The degraded date pits can be mixedwith the conventional ingredients used in animal feeds or the degradeddate pits can replace all or a portion of the conventional animal feed.For example a portion of the corn or soy component can be replaced withan equivalent amount of degraded date pits.

Alternatively the degraded date pit composition may be an additive addedto a pre-formulated feed composition prior to feeding of the feedcomposition to the animal.

The degraded date pit composition can be in the form of a powder.

The degraded date pit composition is present in the feed at aconcentration, or administered in an effective amount which provides anantibacterial effect in the animal. An effective amount refers to anamount of the degraded date pit composition which is effective, uponsingle or multiple dose administration to a subject, in treating orpreventing a bacterial infection beyond that than would have beenexpected in the absence of such a treatment.

The term “antibacterial effect” refers to the ability of the degradeddate pit composition to preventing or delaying the onset of a bacterialinfection in animal, to reducing the microbial load in animals and/or toinhibit the growth or kill bacterium in the animals. Exemplary bacterialpathogens include Escherichia coli, Shigella spp., Salmonella spp.,Camplyobacter spp.

The degraded date pit composition can be present in the feed at aconcentration which provides an equivalent antibacterial effect as thatachieved by the use of conventional antibiotic supplemented feed, suchas a feed supplemented with oxytetracycline.

The present invention is illustrated by the following example and isprovided for exemplification purposes only. The particular examples,materials, amounts, and procedures are not intended to limit the scopeof the invention.

Example

Preparation of Fungus Culture

Trichoderma reesei is grown on potato dextrose agar (PDA) at 25+2° C.for 7 days in the dark.

To confirm that the fungi could degrade the date pits a ground date pitsbased inoculum was prepared by adding half a kilogram of ground datepits and 150 ml of distilled water into 1 liter flasks. The flasks wereautoclaved at 121° C. for 30 min on 3 consecutive days. Under aspeticconditions the ground date pits were then inoculated with 8 agar plugs(6 mm diameter) from actively growing margins of the Trichoderma reesiecolony. The flasks were incubated at 25+2° C. in the dark for threeweeks. The flasks were shaken occasionally to ensure uniformcolonisation of the date pits by the fungi. Colonized ground date pitswhich had been autoclaved twice served as a control.

Small amounts of the colonised and control ground date pits were platedonto PDA to confirm that T. Reesei was present or absent, respectively.

Preparation of Fungi-Degraded Date Pits

Fungi-degraded date pits were produced using a Solid State Degradation(SSD) system inside an incubator.

Date pits of Phonenix dactylifera dates were crushed and grounded usinga medium size mill (Skiold Saeby9300, Denmark) to reduce the size of thepits to about 1 mm in diameter. The ground date pit substrate was mixed,cleaned and sterilized three times at 121° C. for 30 minutes.

Sterilized date pit substrate was added to each cone of the SSD system.The starter culture of the fungus, Trichoderma reesei, prepared on PDAas described above, was added to each cone of the SSD system containingsome of the sterilized date pit substrate. Further fungi cultures andsterilized date pits were added in layers until the volume of fungi anddate pits reached 8 liters per cone. The cones are covered in order toavoid any contamination.

A continuous supply of moistened air is supplied to the SSD system. AnAquafine Ultra violet disinfection system provided the fungi and datepits with disinfected moist air during the incubation period. The SSDsystem is kept within a darkened room at a relative humidity of 90% anda temperature of 30° C. over a 3 week period.

At the end of the 3 week period the process was stopped and the degradeddate pits with the fungi mass was collected and transferred to arefrigerator and kept at 4° C. until use in the feed.

Preparation of Poultry Feed

Six isocaloric-isonitrogenous diets were prepared. The diets wereprepared as described in Table 1 (starter diet) and Table 2 (finisherdiet).

All feed ingredients were ground to a suitable size and mixed in acommercial mixer for 20 minutes. Vitamin and mineral premixes, fish mealand oil were gradually added with continuous mixing the wet mix was thenpass through a commercial mixer for 15 minutes for a homogenousdistribution of the nutrients and particle sizes. The feed containingthe degraded date pits were stored until use at 4° C.

For those feed containing the degraded and non degraded date pits, thedate pit additive was added to the feed alongside the vitamin andmineral premixes. The degraded date pits were prepared as describedabove.

Prevention of Pathogenic Infections

To illustrate the effect of degraded date pit based feed on pathogenicinfections in poultry two hundred, day old chickens were divided intosix groups. Each chicken was housed in separate cleaned and sanitizedPetersime brooding battery cages and kept in a well cleaned anddisinfected poultry house. Water and feed were provided on an ad libitumbasis.

Each group was fed with a different feed composition as follows:

-   1. Group 1 (control)—corn soy diet-   2. Group 2 (control)—corn soy diet+antibiotic added (oxytetracycline    20%, 50 g/100 kg)-   3. Group 3—5% non-degraded date pits corn-soy diet-   4. Group 4—10% non-degraded date pits corn-soy diet-   5. Group 5—5% degraded date pits corn-soy diet-   6. Group 6—10% degraded date pits corn-soy diet

The trial was divided into two periods. Period one (the starter period)started from day 1 to day 21. Period two (the finisher period) startedfrom date 22 to day 33.

During the starter period the chickens were feed a diet having thefollowing composition:

TABLE 1 5% non 10% non 5% 10% degraded degraded degraded degradedIngredient Control date pits date pits date pits date pits Name (kg)(kg) (kg) (kg) (kg) Yellow corn 59.4 53.7 46.6 53.7 46.6 Soybean meal 3230.76 31.25 30.76 31.25 Salt 0.4 0.38 0.38 0.38 0.38 Limestone 1.1 1.051.1 1.05 1.1 Dicalcium 1.56 1.22 1.2 1.22 1.2 phosphate Vitamin and 1 11 1 1 Mineral Premix DL-Methionine 0.24 0.24 0.25 0.24 0.25 Lysine — 0.10.1 0.1 0.1 Corn oil 2 3.35 5.02 3.35 5.02 Fish meal 2.3 3.2 3.1 3.2 3.1Non-Degraded — 5 10 — — Date pits Degraded Date — — — 5 10 pits

During the finisher period the chickens were feed a diet having thefollowing composition:

TABLE 2 5% non 10% non 5% 10% degraded degraded degraded degradedIngredient Control date pits date pits date pits date pits Name (kg)(kg) (kg) (kg) (kg) Yellow corn 64.6 58.25 52.14 58.25 52.14 Soybeanmeal 28.4 27.72 26 27.72 26 Salt 0.42 0.36 0.33 0.36 0.33 Limestone 1.331.22 1.15 1.22 1.15 Dicalcium 1.05 0.95 0.8 0.95 0.8 phosphate Vitaminand 0.2 0.2 0.2 0.2 0.2 Mineral Premix DL-Methionine 0.2 0.2 0.3 0.2 0.3Lysine 0.1 0.1 0.18 0.1 0.18 Corn oil 2.5 4.05 5.9 4.05 5.9 Fish meal1.2 1.95 3 1.95 3 Non-Degraded — 5 10 — — Date pits Degraded Date — — —5 10 pits

At the end of the trial period microbial population densities in drychicken gut tissue were determined for: total bacterial counts,Salmonella spp., Camplylobacter spp., Shigella spp. and Escherichiacoli.

Microbial population densities in log 10 colony-forming units/g drychicken gut were determined by the serial dilution tenfold plate assay.The results are shown in Table 3.

TABLE 3 Population density (mean log₁₀ cfu per g dry gut tissue) TotalSalmo- Campylo- Shigel- Esch- bacterial nella bacter la erichiaTreatments counts spp. spp. spp. coli (1)Control 8.53 ± 2.03 ± 2.74 ±2.12 ± 7.19 ± (without (0.88)^(a) (0.13)^(a) (0.09)^(a) (0.07)^(e)(0.14)^(a) antibiotics) (2) Control 4.21 ± 0.03 ± 0.08 ± 0.53 ± 2.06 ±With (0.14)^(b) (0.03)^(b) (0.05)^(b) (0.06)^(b) (0.18)^(b) antibiotics(3) With 5% 7.07 ± 1.41 ± 2.01 ± 1.90 ± 5.53 ± un-degraded (0.91)^(c)(0.08)^(c) (0.10)^(c) (0.07)^(de) (0.14)^(c) date pits (4) With 10% 6.02± 1.48 ± 0.95 ± 1.67 ± 4.61 ± un-degraded (0.12)^(d) (0.11)^(c)(0.07)^(d) (0.09)^(d) (0.16)^(d) date pits (5) With 5% 4.99 ± 0.61 ±0.10 ± 0.78 ± 3.06 ± degraded date (0.11)^(e) (0.10)^(d) (0.06)^(b)(0.11)^(c) (0.17)^(e) pits (6) With 10% 3.95 ± 0.05 ± 0.06 ± 0.25 ± 2.21± degraded date (0.12)^(b) (0.05)^(b) (0.04)^(b) (0.09)^(a) (0.13)^(b)pits

The values in Table 3 are means of 6 replicates for each treatment andthe values in parentheses are the standard error of the mean. Valueswith the same letter within a column are not significantly (P>0.01)different according to Fisher's Protected LSD Test.

Chicken gut samples from Group 1 (control group without antibioticsadded) had significantly higher (P<0.01) higher total microbialpopulations than the chicken gut samples fed degraded and un-degradeddate pits treatments (group 3, 4, 5, and 6) as well as the control dietwith added tetracyclin antibiotic (group 2).

The estimated total populations of total aerobic bacteria, Escherichiacoli, Salmonella spp., Shigella spp. and Camplyobacter spp. aresignificantly (P<0.01) lower in the samples with degraded andun-degraded date pits (groups 3, 4, 5, and 6) compared to the samplewithout date pits (group 1). There are also significant (P<0.01)decreases in microbial populations by increasing the concentration ofdate pits.

The treatment which included degraded date pits in 10% concentration(group 6) is significantly superior to other treatments in suppressingmicrobial population and is shown to provide a reduction in microbialloads equivalent to that achieved by the use of an antibioticsupplemented feed (group 2).

These results show that the degraded date pits could replaceconventional antibiotics such as, oxytetracycline-20% in the treatmentand prevention of bacterial infections in poultry.

During the trial weight gain and feed intake where measured. The feedconversion ratio (FCR), was calculated. The feed conversion ratio isdefined as the amount of feed (in kg) consumed by the animal to produce1 kg of weight gain. The averaged results per chicken are shown in Table4.

TABLE 4 Feed- Feed- Weight-Gain Weight-Gain Feed-Intake Feed-IntakeConversion Conversion Starter (g) Total (g) Starter (g) Total (g) RatioStarter Ratio Total Treatments (WGS) (WGT) (FIS) (FIT) (FCRS) (FCRT) (1)Control 866 1694.0 1200.6 1437.0 1.387 1.557 (without antibiotics) (2)Control 829.7 1729.3 1167.5 1282.9 1.420 1.423 (with antibiotics) (3)With 923.3 1778.7 1250.1 1488.6 1.353 1.540 5% non- degraded date pits(4) With 900.3 1729.7 1250.9 1439.4 1.387 1.556 10% non- degraded datepits (5) With 835.7 1619 1199.8 1340.5 1.450 1.570 5% degraded date pits(6) With 840 1652.7 1214.6 1387.2 1.450 1.573 10% degraded date pits

The total weight gain from groups 5 and 6 (degraded date pits) were notsignificantly difference (P>0.05) from the group 2 (control group withantibiotics added). There was no significant difference on body weightgain in the poultry when degraded date pits were added to the feedcomposition to treat and prevent bacterial infections.

These results show that the treatment and prevention of bacterialinfections in poultry can be achieved by replacing part of aconventional feed composition with degraded date pits, as an alternativeto using conventional antibiotics, whilst still achieving substantiallythe same weight gains and FCR as achieved when conventional antibioticsare administered.

Further changes can be made within the scope of the invention. Forexample other processes can be used to obtain a degraded date pitcomposition. The degraded date pit compositions can be added to poultryfeeds having a different composition. The degraded date pit compositionscan be administered to other animals.

The invention claimed is:
 1. A method for producing an antibacterialfeed additive for treating a bacterial infection in an animal subject inneed thereof, the method comprising subjecting date pits to solid statedegradation using Trichoderma reesei to form a Trichodermareesei-degraded date pit composition.
 2. The method as claimed in claim1, wherein the bacterial infection is caused by at least one ofSalmonella species, Campylobacter species, Shigella species, andEscherichia species.
 3. The method as claimed in claim 1, wherein theTrichoderma reesei-degraded date pit composition comprisesmannan-oligosaccharides (MOS).
 4. The method as claimed in claim 1,wherein the Trichoderma reesei-degraded date pit composition comprisesfree mannose.
 5. The method as claimed in claim 1, wherein theantibacterial feed additive is in the form of a powder.
 6. A method forpreparing an antibacterial feed composition for use in treating abacterial infection in an animal subject in need thereof, the methodcomprising subjecting date pits to solid state degradation usingTrichoderma reesei to form Trichoderma reesei-de graded date pitcomposition, and mixing the Trichoderma reesei-degraded date pitcomposition with an animal feed composition.
 7. The method as claimed inclaim 6, wherein the bacterial infection is caused by at least one ofSalmonella species, Campylobacter species, Shigella species, andEscherichia species.
 8. The method as claimed in claim 6, wherein theTrichoderma reesei-degraded date pit composition is mixed with theanimal feed composition to achieve a concentration of the Trichodermareesei-degraded date pit composition of between 5-20% w/w relative tothe animal feed composition.
 9. The method as claimed in claim 6,wherein the Trichoderma reesei-degraded date pit composition is mixedwith the animal feed composition to achieve a concentration of theTrichoderma reesei-degraded date pit composition of about 10% w/wrelative to the animal feed composition.
 10. The method as claimed inclaim 6, wherein the animal feed composition comprises at least oneselected from the group consisting of corn, soybean meal, vitamins,minerals, amino acids, corn oil and fish meal.
 11. The method as claimedin claim 6, wherein the animal subject is selected from the groupconsisting of cattle, horses, pigs, goats, and poultry.